Drosophila Enhancer Research Articles

Transcription through enhancers suppresses their activity in Drosophila

BackgroundEnhancer elements determine the level of target gene transcription in a tissue-specific manner, providing for individual patterns of gene expression in different cells. Knowledge of the mechanisms controlling enhancer action is crucial for understanding global regulation of transcription. In particular, enhancers are often localized within transcribed regions of the genome. A number of experiments suggest that transcription can have both positive and negative effects on regulatory elements. In this study, we performed direct tests for the effect of transcription on enhancer activity.ResultsUsing a transgenic reporter system, we investigated the relationship between the presence of pass-through transcription and the activity of Drosophila enhancers controlling the expression of the white and yellow genes. The results show that transcription from different promoters affects the activity of enhancers, counteracting their ability to activate the target genes. As expected, the presence of a transcriptional terminator between the inhibiting promoter and the affected enhancer strongly reduces the suppression. Moreover, transcription leads to dislodging of the Zeste protein that is responsible for the enhancer-dependent regulation of the white gene, suggesting a 'transcription interference’ mechanism for this regulation.ConclusionsOur findings suggest a role for pass-through transcription in negative regulation of enhancer activity.

Enhanced dissection of the regulatory genome

Methods for high-throughput and high-resolution dissection of enhancers in Drosophila are described by two independent groups.

Drosophila SETs Its Sights on Cancer: Trr/MLL3/4 COMPASS-Like Complexes in Development and Disease

Drosophila SETs Its Sights on Cancer: Trr/MLL3/4 COMPASS-Like Complexes in Development and Disease

A Review on EZH2 and its Epigenetic Association with Breast Cancer

Enhancer of zeste homolog2 (EZH2), first identified as homolog of the Drosophila enhancer of zeste gene, is histone H3 lysine methyltransferase (H3K27me3), a component of polycomb group proteins (PRC2) that represses the gene expression by modifying the histones epigenetically, thereby silencing developmental regulatory elements in stem as well as cancer cells leading to repression of early differentiation marker genes. Although the mechanistic approach of involving EZH2 to cancer progression has not yet been clearly deciphered, its invasiveness and metastatic potential has been revealed by significant elevation of its expression in normal breast cancer cells after commencement of which a pre-cancerous state was found in morphologically normal breast cancer cells. The tissue microarray analysis of breast carcinomas has shown that EZH2 to be intimately associated with markers of tumor cell proliferation as well as with aggressive diseases. Till now, no demethylating agents have been recommended for treatment of patients, but an in-vitro study using 3-deazaneplanocin, which reduces histone modifications through methylation by reducing the levels of EZH2, has shown a significant reduction in cell proliferation in breast cancer cells. This further signifies the role of EZH2 as a transcriptional repressor. By analyzing methylation profiles of different subtypes of breast cancers like basal-like, luminal A & B, roles of EZH2 have been established in the development of breast cancers. Crosstalk of EZH2 with other silencing/regulating factors like histone deacetylases and miRNAs, have to be considered for evaluating for progression of cell proliferation in different cancer cells including breast cancer.

Transactivation in Drosophila of human enhancers by human transcription factors involved in congenital heart diseases

Background: The human transcription factors (TFs) GATA4, NKX2.5 and TBX5 form part of the core network necessary to build a human heart and are involved in Congenital Heart Diseases (CHDs). The human natriuretic peptide precursor A (NPPA) and α-myosin heavy chain 6 (MYH6) genes are downstream effectors involved in cardiogenesis that have been demonstrated to be in vitro targets of such TFs. Results: To study the interactions between these human TFs and their target enhancers in vivo, we overexpressed them in the whole Drosophila cardiac tube using the UAS/GAL4 system. We observed that all three TFs up-regulate their natural target enhancers in Drosophila and cause developmental defects when overexpressed in eyes and wings. Conclusions: A strong potential of the present model might be the development of combinatorial and mutational assays to study the interactions between human TFs and their natural target promoters, which are not easily undertaken in tissue culture cells because of the variability in transfection efficiency, especially when multiple constructs are used. Thus, this novel system could be used to determine in vivo the genetic nature of the human mutant forms of these TFs, setting up a powerful tool to unravel the molecular genetic mechanisms that lead to CHDs. Developmental Dynamics 241:190–199, 2012. © 2011 Wiley Periodicals, Inc.Key findingsThe mammalian transcription factors GATA4, Nkx2.5 and TBX5 are able to transactivate the Nppa and Myh6 human enhancers in Drosophila embryos.The endogenous Drosophila homologs of GATA4, Nkx2.5 and TBX5 are not transactivated by GATA4, Nkx2.5 or TBX5, ruling out a possible endogenous regulatory circuit.Overexpression in the mesoderm of GATA4, Nkx2.5 or TBX5 is lethal and overexpression in eyes and wings causes reduced organs.

Modeling the Evolution of Regulatory Elements by Simultaneous Detection and Alignment with Phylogenetic Pair HMMs

The computational detection of regulatory elements in DNA is a difficult but important problem impacting our progress in understanding the complex nature of eukaryotic gene regulation. Attempts to utilize cross-species conservation for this task have been hampered both by evolutionary changes of functional sites and poor performance of general-purpose alignment programs when applied to non-coding sequence. We describe a new and flexible framework for modeling binding site evolution in multiple related genomes, based on phylogenetic pair hidden Markov models which explicitly model the gain and loss of binding sites along a phylogeny. We demonstrate the value of this framework for both the alignment of regulatory regions and the inference of precise binding-site locations within those regions. As the underlying formalism is a stochastic, generative model, it can also be used to simulate the evolution of regulatory elements. Our implementation is scalable in terms of numbers of species and sequence lengths and can produce alignments and binding-site predictions with accuracy rivaling or exceeding current systems that specialize in only alignment or only binding-site prediction. We demonstrate the validity and power of various model components on extensive simulations of realistic sequence data and apply a specific model to study Drosophila enhancers in as many as ten related genomes and in the presence of gain and loss of binding sites. Different models and modeling assumptions can be easily specified, thus providing an invaluable tool for the exploration of biological hypotheses that can drive improvements in our understanding of the mechanisms and evolution of gene regulation.

ZFP423 Coordinates Notch and Bone Morphogenetic Protein Signaling, Selectively Up-regulating Hes5 Gene Expression

Zinc finger protein 423 encodes a 30 Zn-finger transcription factor involved in cerebellar and olfactory development. ZFP423 is a known interactor of SMAD1-SMAD4 and of Collier/Olf-1/EBF proteins, and acts as a modifier of retinoic acid-induced differentiation. In the present article, we show that ZFP423 interacts with the Notch1 intracellular domain in mammalian cell lines and in Xenopus neurula embryos, to activate the expression of the Notch1 target Hes5/ESR1. This effect is antagonized by EBF transcription factors, both in cultured cells and in Xenopus embryos, and amplified in vitro by BMP4, suggesting that ZFP423 acts to integrate BMP and Notch signaling, selectively promoting their convergence onto the Hes5 gene promoter.

Gene Regulation: The Cohesin Ring Connects Developmental Highways

How does cohesin regulate gene expression and development independently of its roles in sister chromatid cohesion and chromosome segregation? Recent studies show that cohesin, through multiple mechanisms, directly controls transcription of genes that regulate morphogenesis, differentiation, cell proliferation and pluripotency.

Thermodynamics-Based Models of Transcriptional Regulation by Enhancers: The Roles of Synergistic Activation, Cooperative Binding and Short-Range Repression

Quantitative models of cis-regulatory activity have the potential to improve our mechanistic understanding of transcriptional regulation. However, the few models available today have been based on simplistic assumptions about the sequences being modeled, or heuristic approximations of the underlying regulatory mechanisms. We have developed a thermodynamics-based model to predict gene expression driven by any DNA sequence, as a function of transcription factor concentrations and their DNA-binding specificities. It uses statistical thermodynamics theory to model not only protein-DNA interaction, but also the effect of DNA-bound activators and repressors on gene expression. In addition, the model incorporates mechanistic features such as synergistic effect of multiple activators, short range repression, and cooperativity in transcription factor-DNA binding, allowing us to systematically evaluate the significance of these features in the context of available expression data. Using this model on segmentation-related enhancers in Drosophila, we find that transcriptional synergy due to simultaneous action of multiple activators helps explain the data beyond what can be explained by cooperative DNA-binding alone. We find clear support for the phenomenon of short-range repression, where repressors do not directly interact with the basal transcriptional machinery. We also find that the binding sites contributing to an enhancer's function may not be conserved during evolution, and a noticeable fraction of these undergo lineage-specific changes. Our implementation of the model, called GEMSTAT, is the first publicly available program for simultaneously modeling the regulatory activities of a given set of sequences.

An alignment-free method to identify candidate orthologous enhancers in multiple Drosophila genomes

Evolutionarily conserved non-coding genomic sequences represent a potentially rich source for the discovery of gene regulatory region such as transcriptional enhancers. However, detecting orthologous enhancers using alignment-based methods in higher eukaryotic genomes is particularly challenging, as regulatory regions can undergo considerable sequence changes while maintaining their functionality. We have developed an alignment-free method which identifies conserved enhancers in multiple diverged species. Our method is based on similarity metrics between two sequences based on the co-occurrence of sequence patterns regardless of their order and orientation, thus tolerating sequence changes observed in non-coding evolution. We show that our method is highly successful in detecting orthologous enhancers in distantly related species without requiring additional information such as knowledge about transcription factors involved, or predicted binding sites. By estimating the significance of similarity scores, we are able to discriminate experimentally validated functional enhancers from seemingly equally conserved candidates without function. We demonstrate the effectiveness of this approach on a wide range of enhancers in Drosophila, and also present encouraging results to detect conserved functional regions across large evolutionary distances. Our work provides encouraging steps on the way to ab initio unbiased enhancer prediction to complement ongoing experimental efforts. The software, data and the results used in this article are available at http://www.genome.duke.edu/labs/ohler/research/transcription/fly_enhancer/.

Evolution of a genomic regulatory domain: The role of gene co-option and gene duplication in the Enhancer of split complex

The Drosophila Enhancer of split complex [E(spl)-C] is a remarkable complex of genes many of which are effectors or modulators of Notch signaling. The complex contains different classes of genes including four bearded genes and seven basic helix-loop-helix (bHLH) genes. We examined the evolution of this unusual complex by identifying bearded and bHLH genes in the genome sequences of Arthropods. We find that a four-gene E(spl)-C, containing three bHLH genes and one bearded gene, is an ancient component of the genomes of Crustacea and Insects. The complex is well conserved in insects but is highly modified in Drosophila, where two of the ancestral genes of the complex are missing, and the remaining two have been duplicated multiple times. Through examining the expression of E(spl)-C genes in honeybees, aphids, and Drosophila, we determined that the complex ancestrally had a role in Notch signaling. The expression patterns of genes found inserted into the complex in some insects, or that of ancestral E(spl)-C genes that have moved out of the complex, imply that the E(spl)-C is a genomic domain regulated as a whole by Notch signaling. We hypothesize that the E(spl)-C is a Notch-regulated genomic domain conserved in Arthropod genomes for around 420 million years. We discuss the consequence of this conserved domain for the recruitment of novel genes into the Notch signaling cascade.

Evolutionary Mirages: Selection on Binding Site Composition Creates the Illusion of Conserved Grammars in Drosophila Enhancers

The clustering of transcription factor binding sites in developmental enhancers and the apparent preferential conservation of clustered sites have been widely interpreted as proof that spatially constrained physical interactions between transcription factors are required for regulatory function. However, we show here that selection on the composition of enhancers alone, and not their internal structure, leads to the accumulation of clustered sites with evolutionary dynamics that suggest they are preferentially conserved. We simulated the evolution of idealized enhancers from Drosophila melanogaster constrained to contain only a minimum number of binding sites for one or more factors. Under this constraint, mutations that destroy an existing binding site are tolerated only if a compensating site has emerged elsewhere in the enhancer. Overlapping sites, such as those frequently observed for the activator Bicoid and repressor Krüppel, had significantly longer evolutionary half-lives than isolated sites for the same factors. This leads to a substantially higher density of overlapping sites than expected by chance and the appearance that such sites are preferentially conserved. Because D. melanogaster (like many other species) has a bias for deletions over insertions, sites tended to become closer together over time, leading to an overall clustering of sites in the absence of any selection for clustered sites. Since this effect is strongest for the oldest sites, clustered sites also incorrectly appear to be preferentially conserved. Following speciation, sites tend to be closer together in all descendent species than in their common ancestors, violating the common assumption that shared features of species' genomes reflect their ancestral state. Finally, we show that selection on binding site composition alone recapitulates the observed number of overlapping and closely neighboring sites in real D. melanogaster enhancers. Thus, this study calls into question the common practice of inferring “cis-regulatory grammars” from the organization and evolutionary dynamics of developmental enhancers.

Regulation of the Drosophila Enhancer of split and invected-engrailed Gene Complexes by Sister Chromatid Cohesion Proteins

The cohesin protein complex was first recognized for holding sister chromatids together and ensuring proper chromosome segregation. Cohesin also regulates gene expression, but the mechanisms are unknown. Cohesin associates preferentially with active genes, and is generally absent from regions in which histone H3 is methylated by the Enhancer of zeste [E(z)] Polycomb group silencing protein. Here we show that transcription is hypersensitive to cohesin levels in two exceptional cases where cohesin and the E(z)-mediated histone methylation simultaneously coat the entire Enhancer of split and invected-engrailed gene complexes in cells derived from Drosophila central nervous system. These gene complexes are modestly transcribed, and produce seven of the twelve transcripts that increase the most with cohesin knockdown genome-wide. Cohesin mutations alter eye development in the same manner as increased Enhancer of split activity, suggesting that similar regulation occurs in vivo. We propose that cohesin helps restrain transcription of these gene complexes, and that deregulation of similarly cohesin-hypersensitive genes may underlie developmental deficits in Cornelia de Lange syndrome.

Functional Conservation of Asxl2, a Murine Homolog for the Drosophila Enhancer of Trithorax and Polycomb Group Gene Asx

BackgroundPolycomb-group (PcG) and trithorax-group (trxG) proteins regulate histone methylation to establish repressive and active chromatin configurations at target loci, respectively. These chromatin configurations are passed on from mother to daughter cells, thereby causing heritable changes in gene expression. The activities of PcG and trxG proteins are regulated by a special class of proteins known as Enhancers of trithorax and Polycomb (ETP). The Drosophila gene Additional sex combs (Asx) encodes an ETP protein and mutations in Asx enhance both PcG and trxG mutant phenotypes. The mouse and human genomes each contain three Asx homologues, Asx-like 1, 2, and 3. In order to understand the functions of mammalian Asx-like (Asxl) proteins, we generated an Asxl2 mutant mouse from a gene-trap ES cell line.Methodology/Principal FindingsWe show that the Asxl2 gene trap is expressed at high levels in specific tissues including the heart, the axial skeleton, the neocortex, the retina, spermatogonia and developing oocytes. The gene trap mutation is partially embryonic lethal and approximately half of homozygous animals die before birth. Homozygotes that survive embryogenesis are significantly smaller than controls and have a shortened life span. Asxl2−/− mice display both posterior transformations and anterior transformation in the axial skeleton, suggesting that the loss of Asxl2 disrupts the activities of both PcG and trxG proteins. The PcG-associated histone modification, trimethylation of histone H3 lysine 27, is reduced in Asxl2−/− heart. Necropsy and histological analysis show that mutant mice have enlarged hearts and may have impaired heart function.Conclusions/SignificanceOur results suggest that murine Asxl2 has conserved ETP function and plays dual roles in the promotion of PcG and trxG activity. We have also revealed an unexpected role for Asxl2 in the heart, suggesting that the PcG/trxG system may be involved in the regulation of cardiac function.

Using a combination of SAR element and enhancer to increase the expression level of hybrid genes in transgenic animals

A regulatory element consisting of a MAR element sequence of Drosophila heat shock genes and enhancer from a long terminal repeat of the mouse mammary tumor virus is created for studying the possibility of increasing the expression level of hybrid genes in transgenic animals. Two lines of transgenic mice containing the described regulatory element and hybrid gene intended for expressing human beta interferon in milk of transgenic animals are obtained by the method of coinjection into the pronuclei of murine zygotes. It is shown that the use of the given regulatory element doesn’t lead to an increase of activity of human beta interferon in milk compared with transgenic rabbits containing only the hybrid gene for expressing beta interferon. Furthermore, a decrease of tissue-specific expression of beta interferon is observed with the use of the regulatory element.

Sepsid even-skipped enhancers are functionally conserved in Drosophila despite lack of sequence conservation.

The gene expression pattern specified by an animal regulatory sequence is generally viewed as arising from the particular arrangement of transcription factor binding sites it contains. However, we demonstrate here that regulatory sequences whose binding sites have been almost completely rearranged can still produce identical outputs. We sequenced the even-skipped locus from six species of scavenger flies (Sepsidae) that are highly diverged from the model species Drosophila melanogaster, but share its basic patterns of developmental gene expression. Although there is little sequence similarity between the sepsid eve enhancers and their well-characterized D. melanogaster counterparts, the sepsid and Drosophila enhancers drive nearly identical expression patterns in transgenic D. melanogaster embryos. We conclude that the molecular machinery that connects regulatory sequences to the transcription apparatus is more flexible than previously appreciated. In exploring this diverse collection of sequences to identify the shared features that account for their similar functions, we found a small number of short (20–30 bp) sequences nearly perfectly conserved among the species. These highly conserved sequences are strongly enriched for pairs of overlapping or adjacent binding sites. Together, these observations suggest that the local arrangement of binding sites relative to each other is more important than their overall arrangement into larger units of cis-regulatory function.

Identification and characterization of human HESL, rat Hesl and rainbow trout hesl genes in silico.

Activation of Notch signaling pathway leads to nuclear translocation of Notch intracellular domain (NIC), and transcriptional activation of target genes through the interaction between CSL proteins (RBPSUH or RBPSUHL) and NIC. HES1, HES5, HEY1 and HEY2 are Notch target genes. Mammalian HES/HEY family proteins as well as Drosophila Hairy and Enhancer of split are implicated in the cell fate determination. We have previously identified and characterized human HES2, HES3 and HES5 genes. Here, we identified and characterized human HES-like (HESL), rat Fesl, and rainbow trout fesl genes by using bioinformatics. Human HESL gene, located within AC093824.3 genome sequence, was mapped to human chromosome 4q35.1 between ACSL1 and SLC25A4 genes. Rat Fesl gene, located within AC111303.4 genome sequence, was mapped to rat chromosome 16q11. EST BX875070.2 was the representative rainbow trout fesl cDNA. HESL-ACSL1 locus was conserved among human, rat, mouse, and zebrafish genomes. Human HESL (241 aa) showed 92.9% total-amino-acid identity with rat Hesl (240 aa), 92.1% total-amino-acid identity with mouse Hesl (240 aa), and 68.0% total-amino-acid identity with rainbow trout hesl (235 aa). HESL orthologs consist of basic Helix-loop-helix (bHLH) domain and ORANGE domain. C-terminal region of HESL orthologs were divergent from that of HES, HEY, and DEC homologs. Phylogenetic analyses revealed that bHLH transcription factors with ORANGE domain were classified into the following three groups: (group I) HES1, HES2, HES4 and HES6 orthologs; (group II) HESL, HES5, HES7, HEY1, HEY2 and HEYL orthologs; (group III) HES3, DEC1/BHLHB2 and DEC2/BHLHB3 orthologs.

The Polycomb-group protein ENX-2 interacts with ZAP-70

Human ENX-2 is a homologue of Drosophila Enhancer of zeste, which is a member of Polycomb-group proteins regulating the expression of homeotic genes as chromatin-associated proteins. In this study, we demonstrate that ENX-2 plays an important role as a signaling molecule involved in T cell receptor-mediated signaling pathway. In immunoprecipitation experiments, ENX-2 and zeta associated protein-70 (ZAP-70) were co-precipitated from T cell lysate. When probed with an anti-phospho-tyrosine antibody, ENX-2 was found to be phosphorylated on tyrosine. On the other hand, ENX-2 was not phosphorylated on tyrosine in the mutant Jurkat cell, J.Cam1.6 lacking the activity of lymphocyte protein tyrosine kinase p56 lck. The interaction between ENX-2 and ZAP-70 was abolished in the mutant cell. Furthermore, in-vitro kinase assay using purified p56 lck demonstrated that ENX-2 became tyrosine phosphorylated by this kinase. These findings show that the phosphorylation of ENX-2 is responsible for the interaction between ENX-2 and ZAP-70.

Cysteine-rich region of X-Serrate-1 is required for activation of Notch signaling in Xenopus primary neurogenesis.

The Notch family genes encode single-pass transmembrane proteins which function in a variety of cell fate specifications in invertebrates and vertebrates. In Xenopus primary neurogenesis, the Notch ligands, X-Delta-1 and X-Serrate-1, mediate Notch signaling and regulate cell differentiation. In the present study, we examined the role of the Serrate-specific cysteine-rich (CR) region in the primary neurogenesis of Xenopus embryos. The ligand constructs containing the DSL (Delta/Serrate/Lag-2) domain in the extracellular region caused a reduction in primary neurons, whereas the DSL-deleted form of X-Delta-1 resulted in the overproduction of primary neurons. However, the DSL-deleted form of X-Serrate-1 or the construct containing only the CR region in the extracellular domain (SerCR) reduced the number of primary neurons. In contrast, the CR-deleted form of X-Serrate-1 (SerACR) lost activity as a Notch ligand, regardless of the presence of the DSL domain within the extracellular domain. Overexpression of X-Delta-1 and X-Serrate-1 strongly induced the expression of Xenopus ESR-1 (XESR-1), a gene related to Drosophila Enhancer of split. SerCR alone also moderately induced the expression of XESR-1, but the SerACR form did not induce this expression. Co-injection of X-Notch-1deltaICD, which deletes the intracellular domain (ICD), with SerCR suppressed a neurogenic phenotype, although co-injection of X-Su(H)1DBM with SerCR did not, indicating that SerCR affects primary neurogenesis through the Notch/Su(H) pathway. These results suggestthatthe CR region of Xenopus Serrate is required for the activation of Notch signaling and cell fate specification in primary neurogenesis.

Drosophila Enhancer of Zeste/ESC Complexes Have a Histone H3 Methyltransferase Activity that Marks Chromosomal Polycomb Sites

Enhancer of Zeste is a Polycomb Group protein essential for the establishment and maintenance of repression of homeotic and other genes. In the early embryo it is found in a complex that includes ESC and is recruited to Polycomb Response Elements. We show that this complex contains a methyltransferase activity that methylates lysine 9 and lysine 27 of histone H3, but the activity is lost when the E(Z) SET domain is mutated. The lysine 9 position is trimethylated and this mark is closely associated with Polycomb binding sites on polytene chromosomes but is also found in centric heterochromatin, chromosome 4, and telomeric sites. Histone H3 methylated in vitro by the E(Z)/ESC complex binds specifically to Polycomb protein.